Search

Interleukin 5 (IL-5) is a critical cytokine for the maturation of eosinophil precursors to eosinophils in the bone marrow and those eosinophils then accumulate in the lungs during asthma. We have studied anti IL-5 antibodies on allergic responses in mice, guinea pigs and monkeys and are extending this experiment into humans with a humanized antibody. In a monkey model of pulmonary inflammation and airway hyperreactivity, we found that the TRFK-5 antibody blocked both responses for three months following a single dose of 0.3 mg/kg, i.v. This antibody also blocked lung eosinophilia in mice by inhibiting release from the bone marrow. To facilitate multiple dosing and to reduce immunogenicity in humans, we prepared Sch 55700, a humanized antibody against IL-5. Sch 55700 was also active against lung eosinophilia in allergic monkeys and mice and against pulmonary eosinophilia and airway hyperresponsiveness in guinea pigs. Furthermore, as opposed to steroids, Sch 55700 did not cause immunosuppression in guinea pigs. Studies with this antibody in humans will be critical to establishing the therapeutic potential of IL-5 inhibition.

Thymic stromal cells (TSCs) are critical regulators of T cell tolerance, but their basic biology has remained under-characterized because they are relatively rare and difficult to isolate. Recent work has revealed that constitutive autophagy in TSCs is required for self-antigen presentation and central T cell tolerance induction; however, the mechanisms regulating constitutive autophagy in TSCs are not well understood. Hydrogen peroxide has been shown to increase autophagy flux in other tissues, and we previously identified conspicuously low expression of the hydrogen peroxide-quenching enzyme catalase in TSCs. We investigated whether the redox status of TSCs established by low catalase expression regulates their basal autophagy levels and their capacity to impose central T cell tolerance. Transgenic overexpression of catalase diminished autophagy in TSCs and impaired thymocyte clonal deletion, concomitant with increased frequencies of spontaneous lymphocytic infiltrates in lung and liver and of serum antinuclear antigen reactivity. Effects on clonal deletion and autoimmune indicators were diminished in catalase transgenic mice when autophagy was rescued by expression of the Becn1 F121A/F121A knock-in allele. These results suggest a metabolic mechanism by which the redox status of TSCs may regulate central T cell tolerance.

SMYD2 is a lysine methyltransferase that catalyzes the monomethylation of several protein substrates including p53. SMYD2 is overexpressed in a significant percentage of esophageal squamous primary carcinomas, and that overexpression correlates with poor patient survival. However, the mechanism(s) by which SMYD2 promotes oncogenesis is not understood. A small molecule probe for SMYD2 would allow for the pharmacological dissection of this biology. In this report, we disclose LLY-507, a cell-active, potent small molecule inhibitor of SMYD2. LLY-507 is >100-fold selective for SMYD2 over a broad range of methyltransferase and non-methyltransferase targets. A 1.63-Å resolution crystal structure of SMYD2 in complex with LLY-507 shows the inhibitor binding in the substrate peptide binding pocket. LLY-507 is active in cells as measured by reduction of SMYD2-induced monomethylation of p53 Lys(370) at submicromolar concentrations. We used LLY-507 to further test other potential roles of SMYD2. Mass spectrometry-based proteomics showed that cellular global histone methylation levels were not significantly affected by SMYD2 inhibition with LLY-507, and subcellular fractionation studies indicate that SMYD2 is primarily cytoplasmic, suggesting that SMYD2 targets a very small subset of histones at specific chromatin loci and/or non-histone substrates. Breast and liver cancers were identified through in silico data mining as tumor types that display amplification and/or overexpression of SMYD2. LLY-507 inhibited the proliferation of several esophageal, liver, and breast cancer cell lines in a dose-dependent manner. These findings suggest that LLY-507 serves as a valuable chemical probe to aid in the dissection of SMYD2 function in cancer and other biological processes., (© 2015 by The American Society for Biochemistry and Molecular Biology, Inc.)

Protein arginine methyltransferases (PRMTs) play important roles in several cellular processes, including signaling, gene regulation, and transport of proteins and nucleic acids, to impact growth, differentiation, proliferation, and development. PRMT5 symmetrically di-methylates the two-terminal ω-guanidino nitrogens of arginine residues on substrate proteins. PRMT5 acts as part of a multimeric complex in concert with a variety of partner proteins that regulate its function and specificity. A core component of these complexes is the WD40 protein MEP50/WDR77/p44, which mediates interactions with binding partners and substrates. We have determined the crystal structure of human PRMT5 in complex with MEP50 (methylosome protein 50), bound to an S-adenosylmethionine analog and a peptide substrate derived from histone H4. The structure of the surprising hetero-octameric complex reveals the close interaction between the seven-bladed β-propeller MEP50 and the N-terminal domain of PRMT5, and delineates the structural elements of substrate recognition.

Adenine deaminase (ADE) from the amidohydrolase superfamily (AHS) of enzymes catalyzes the conversion of adenine to hypoxanthine and ammonia. Enzyme isolated from Escherichia coli was largely inactive toward the deamination of adenine. Molecular weight determinations by mass spectrometry provided evidence that multiple histidine and methionine residues were oxygenated. When iron was sequestered with a metal chelator and the growth medium supplemented with Mn(2+) before induction, the post-translational modifications disappeared. Enzyme expressed and purified under these conditions was substantially more active for adenine deamination. Apo-enzyme was prepared and reconstituted with two equivalents of FeSO(4). Inductively coupled plasma mass spectrometry and Mössbauer spectroscopy demonstrated that this protein contained two high-spin ferrous ions per monomer of ADE. In addition to the adenine deaminase activity, [Fe(II) /Fe(II) ]-ADE catalyzed the conversion of H(2)O(2) to O(2) and H(2)O. The values of k(cat) and k(cat)/K(m) for the catalase activity are 200 s(-1) and 2.4 × 10(4) M(-1) s(-1), respectively. [Fe(II)/Fe(II)]-ADE underwent more than 100 turnovers with H(2)O(2) before the enzyme was inactivated due to oxygenation of histidine residues critical for metal binding. The iron in the inactive enzyme was high-spin ferric with g(ave) = 4.3 EPR signal and no evidence of anti-ferromagnetic spin-coupling. A model is proposed for the disproportionation of H(2)O(2) by [Fe(II)/Fe(II)]-ADE that involves the cycling of the binuclear metal center between the di-ferric and di-ferrous oxidation states. Oxygenation of active site residues occurs via release of hydroxyl radicals. These findings represent the first report of redox reaction catalysis by any member of the AHS., (Copyright © 2011 The Protein Society.)

The SET and MYND Domain (SMYD) proteins comprise a unique family of multi-domain SET histone methyltransferases that are implicated in human cancer progression. Here we report an analysis of the crystal structure of the full length human SMYD3 in a complex with an analog of the S-adenosyl methionine (SAM) methyl donor cofactor. The structure revealed an overall compact architecture in which the "split-SET" domain adopts a canonical SET domain fold and closely assembles with a Zn-binding MYND domain and a C-terminal superhelical 9 α-helical bundle similar to that observed for the mouse SMYD1 structure. Together, these structurally interlocked domains impose a highly confined binding pocket for histone substrates, suggesting a regulated mechanism for its enzymatic activity. Our mutational and biochemical analyses confirm regulatory roles of the unique structural elements both inside and outside the core SET domain and establish a previously undetected preference for trimethylation of H4K20.

Per-Arnt-Sim (PAS) domain-containing protein kinase (PASK) is an evolutionary conserved protein kinase that coordinates cellular metabolism with metabolic demand in yeast and mammals. The molecular mechanisms underlying PASK regulation, however, remain unknown. Herein, we describe a crystal structure of the kinase domain of human PASK, which provides insights into the regulatory mechanisms governing catalysis. We show that the kinase domain adopts an active conformation and has catalytic activity in vivo and in vitro in the absence of activation loop phosphorylation. Using site-directed mutagenesis and structural comparison with active and inactive kinases, we identified several key structural features in PASK that enable activation loop phosphorylation-independent activity. Finally, we used combinatorial peptide library screening to determine that PASK prefers basic residues at the P-3 and P-5 positions in substrate peptides. Our results describe the key features of the PASK structure and how those features are important for PASK activity and substrate selection.

Misfolding and degradation of CFTR is the cause of disease in patients with the most prevalent CFTR mutation, an in-frame deletion of phenylalanine (F508del), located in the first nucleotide-binding domain of human CFTR (hNBD1). Studies of (F508del)CFTR cellular folding suggest that both intra- and inter-domain folding is impaired. (F508del)CFTR is a temperature-sensitive mutant, that is, lowering growth temperature, improves both export, and plasma membrane residence times. Yet, paradoxically, F508del does not alter the fold of isolated hNBD1 nor did it seem to perturb its unfolding transition in previous isothermal chemical denaturation studies. We therefore studied the in vitro thermal unfolding of matched hNBD1 constructs ±F508del to shed light on the defective folding mechanism and the basis for the thermal instability of (F508del)CFTR. Using primarily differential scanning calorimetry (DSC) and circular dichroism, we show for all hNBD1 pairs studied, that F508del lowers the unfolding transition temperature (T(m)) by 6-7°C and that unfolding occurs via a kinetically-controlled, irreversible transition in isolated monomers. A thermal unfolding mechanism is derived from nonlinear least squares fitting of comprehensive DSC data sets. All data are consistent with a simple three-state thermal unfolding mechanism for hNBD1 ± F508del: N(±MgATP) <==> I(T)(±MgATP) → A(T) → (A(T))(n). The equilibrium unfolding to intermediate, I(T), is followed by the rate-determining, irreversible formation of a partially folded, aggregation-prone, monomeric state, A(T), for which aggregation to (A(T))(n) and further unfolding occur with no detectable heat change. Fitted parameters indicate that F508del thermodynamically destabilizes the native state, N, and accelerates the formation of A(T).

Upon removal of the regulatory insert (RI), the first nucleotide binding domain (NBD1) of human cystic fibrosis transmembrane conductance regulator (CFTR) can be heterologously expressed and purified in a form that remains stable without solubilizing mutations, stabilizing agents or the regulatory extension (RE). This protein, NBD1 387-646(Delta405-436), crystallizes as a homodimer with a head-to-tail association equivalent to the active conformation observed for NBDs from symmetric ATP transporters. The 1.7-A resolution X-ray structure shows how ATP occupies the signature LSGGQ half-site in CFTR NBD1. The DeltaF508 version of this protein also crystallizes as a homodimer and differs from the wild-type structure only in the vicinity of the disease-causing F508 deletion. A slightly longer construct crystallizes as a monomer. Comparisons of the homodimer structure with this and previously published monomeric structures show that the main effect of ATP binding at the signature site is to order the residues immediately preceding the signature sequence, residues 542-547, in a conformation compatible with nucleotide binding. These residues likely interact with a transmembrane domain intracellular loop in the full-length CFTR channel. The experiments described here show that removing the RI from NBD1 converts it into a well-behaved protein amenable to biophysical studies yielding deeper insights into CFTR function.

The MET receptor tyrosine kinase has emerged as an important target for the development of novel cancer therapeutics. Activation of MET by mutation or gene amplification has been linked to kidney, gastric, and lung cancers. In other cancers, such as glioblastoma, autocrine activation of MET has been demonstrated. Several classes of ATP-competitive inhibitor have been described, which inhibit MET but also other kinases. Here, we describe SGX523, a novel, ATP-competitive kinase inhibitor remarkable for its exquisite selectivity for MET. SGX523 potently inhibited MET with an IC50 of 4 nmol/L and is >1,000-fold selective versus the >200-fold selectivity of other protein kinases tested in biochemical assays. Crystallographic study revealed that SGX523 stabilizes MET in a unique inactive conformation that is inaccessible to other protein kinases, suggesting an explanation for the selectivity. SGX523 inhibited MET-mediated signaling, cell proliferation, and cell migration at nanomolar concentrations but had no effect on signaling dependent on other protein kinases, including the closely related RON, even at micromolar concentrations. SGX523 inhibition of MET in vivo was associated with the dose-dependent inhibition of growth of tumor xenografts derived from human glioblastoma and lung and gastric cancers, confirming the dependence of these tumors on MET catalytic activity. Our results show that SGX523 is the most selective inhibitor of MET catalytic activity described to date and is thus a useful tool to investigate the role of MET kinase in cancer without the confounding effects of promiscuous protein kinase inhibition.

Phase II of the Protein Structure Initiative, funded by the NIH NIGMS (National Institute of General Medical Sciences), is a 5-year effort to determine thousands of protein structures. The New York SGX Research Center for Structural Genomics (NYSGXRC) is one of the four large-scale production centers tasked with determining 100-200 structures annually. Almost all protein production is carried out using the high throughput structural biology platform at SGX Pharmaceuticals (SGX), which supplies 120 or more ultrapure proteins per month for NYSGXRC crystallization and structure determination activities. Protocols for PCR, cloning, expression/solubility testing, fermentation, purification, and crystallization are described. General protocols and detailed experimental results for each target are updated weekly at the public PepcDB website (pepcdb.pdb.org/), and all NYSGXRC clones should be available in 2008 through the PlasmID resource operated by the Harvard Institute of Proteomics.

The New York SGX Research Center for Structural Genomics (NYSGXRC) of the NIGMS Protein Structure Initiative (PSI) has applied its high-throughput X-ray crystallographic structure determination platform to systematic studies of all human protein phosphatases and protein phosphatases from biomedically-relevant pathogens. To date, the NYSGXRC has determined structures of 21 distinct protein phosphatases: 14 from human, 2 from mouse, 2 from the pathogen Toxoplasma gondii, 1 from Trypanosoma brucei, the parasite responsible for African sleeping sickness, and 2 from the principal mosquito vector of malaria in Africa, Anopheles gambiae. These structures provide insights into both normal and pathophysiologic processes, including transcriptional regulation, regulation of major signaling pathways, neural development, and type 1 diabetes. In conjunction with the contributions of other international structural genomics consortia, these efforts promise to provide an unprecedented database and materials repository for structure-guided experimental and computational discovery of inhibitors for all classes of protein phosphatases.

The targets of the Structural GenomiX (SGX) bacterial genomics project were proteins conserved in multiple prokaryotic organisms with no obvious sequence homolog in the Protein Data Bank of known structures. The outcome of this work was 80 structures, covering 60 unique sequences and 49 different genes. Experimental phase determination from proteins incorporating Se-Met was carried out for 45 structures with most of the remainder solved by molecular replacement using members of the experimentally phased set as search models. An automated tool was developed to deposit these structures in the Protein Data Bank, along with the associated X-ray diffraction data (including refined experimental phases) and experimentally confirmed sequences. BLAST comparisons of the SGX structures with structures that had appeared in the Protein Data Bank over the intervening 3.5 years since the SGX target list had been compiled identified homologs for 49 of the 60 unique sequences represented by the SGX structures. This result indicates that, for bacterial structures that are relatively easy to express, purify, and crystallize, the structural coverage of gene space is proceeding rapidly. More distant sequence-structure relationships between the SGX and PDB structures were investigated using PDB-BLAST and Combinatorial Extension (CE). Only one structure, SufD, has a truly unique topology compared to all folds in the PDB., (Copyright 2005 Wiley-Liss, Inc.)

Cystic fibrosis is caused by defects in the cystic fibrosis transmembrane conductance regulator (CFTR), commonly the deletion of residue Phe-508 (DeltaF508) in the first nucleotide-binding domain (NBD1), which results in a severe reduction in the population of functional channels at the epithelial cell surface. Previous studies employing incomplete NBD1 domains have attributed this to aberrant folding of DeltaF508 NBD1. We report structural and biophysical studies on complete human NBD1 domains, which fail to demonstrate significant changes of in vitro stability or folding kinetics in the presence or absence of the DeltaF508 mutation. Crystal structures show minimal changes in protein conformation but substantial changes in local surface topography at the site of the mutation, which is located in the region of NBD1 believed to interact with the first membrane spanning domain of CFTR. These results raise the possibility that the primary effect of DeltaF508 is a disruption of proper interdomain interactions at this site in CFTR rather than interference with the folding of NBD1. Interestingly, increases in the stability of NBD1 constructs are observed upon introduction of second-site mutations that suppress the trafficking defect caused by the DeltaF508 mutation, suggesting that these suppressors might function indirectly by improving the folding efficiency of NBD1 in the context of the full-length protein. The human NBD1 structures also solidify the understanding of CFTR regulation by showing that its two protein segments that can be phosphorylated both adopt multiple conformations that modulate access to the ATPase active site and functional interdomain interfaces.

Structural GenomiX, Inc. (SGX), four New York area institutions, and two University of California schools have formed the New York Structural GenomiX Research Consortium (NYSGXRC), an industrial/academic Research Consortium that exploits individual core competencies to support all aspects of the NIH-NIGMS funded Protein Structure Initiative (PSI), including protein family classification and target selection, generation of protein for biophysical analyses, sample preparation for structural studies, structure determination and analyses, and dissemination of results. At the end of the PSI Pilot Study Phase (PSI-1), the NYSGXRC will be capable of producing 100-200 experimentally determined protein structures annually. All Consortium activities can be scaled to increase production capacity significantly during the Production Phase of the PSI (PSI-2). The Consortium utilizes both centralized and de-centralized production teams with clearly defined deliverables and hand-off procedures that are supported by a web-based target/sample tracking system (SGX Laboratory Information Data Management System, LIMS, and NYSGXRC Internal Consortium Experimental Database, ICE-DB). Consortium management is provided by an Executive Committee, which is composed of the PI and all Co-PIs. Progress to date is tracked on a publicly available Consortium web site (http://www.nysgxrc.org) and all DNA/protein reagents and experimental protocols are distributed freely from the New York City Area institutions. In addition to meeting the requirements of the Pilot Study Phase and preparing for the Production Phase of the PSI, the NYSGXRC aims to develop modular technologies that are transferable to structural biology laboratories in both academe and industry. The NYSGXRC PI and Co-PIs intend the PSI to have a transforming effect on the disciplines of X-ray crystallography and NMR spectroscopy of biological macromolecules. Working with other PSI-funded Centers, the NYSGXRC seeks to create the structural biology laboratory of the future. Herein, we present an overview of the organization of the NYSGXRC and describe progress toward development of a high-throughput Gene-->Structure platform. An analysis of current and projected consortium metrics reflects progress to date and delineates opportunities for further technology development.

Cystic fibrosis transmembrane conductance regulator (CFTR) is an ATP-binding cassette (ABC) transporter that functions as a chloride channel. Nucleotide-binding domain 1 (NBD1), one of two ABC domains in CFTR, also contains sites for the predominant CF-causing mutation and, potentially, for regulatory phosphorylation. We have determined crystal structures for mouse NBD1 in unliganded, ADP- and ATP-bound states, with and without phosphorylation. This NBD1 differs from typical ABC domains in having added regulatory segments, a foreshortened subdomain interconnection, and an unusual nucleotide conformation. Moreover, isolated NBD1 has undetectable ATPase activity and its structure is essentially the same independent of ligand state. Phe508, which is commonly deleted in CF, is exposed at a putative NBD1-transmembrane interface. Our results are consistent with a CFTR mechanism, whereby channel gating occurs through ATP binding in an NBD1-NBD2 nucleotide sandwich that forms upon displacement of NBD1 regulatory segments.

Exon trapping and cDNA selection procedures were used to search for novel genes at human chromosome 11p13, a region previously associated with loss of heterozygosity in epithelial carcinomas. Using these approaches, we found the ESE-2 and ESE-3 genes, coding for ETS domain-containing transcription factors. These genes lie in close proximity to the catalase gene within a approximately 200-kilobase genomic interval. ESE-3 mRNA is widely expressed in human tissues with high epithelial content, and immunohistochemical analysis with a newly generated monoclonal antibody revealed that ESE-3 is a nuclear protein expressed exclusively in differentiated epithelial cells and that it is absent in the epithelial carcinomas tested. In transient transfections, ESE-3 behaves as a repressor of the Ras- or phorbol ester-induced transcriptional activation of a subset of promoters that contain ETS and AP-1 binding sites. ESE-3-mediated repression is sequence- and context-dependent and depends both on the presence of high affinity ESE-3 binding sites in combination with AP-1 cis-elements and the arrangement of these sites within a given promoter. We propose that ESE-3 might be an important determinant in the control of epithelial differentiation, as a modulator of the nuclear response to mitogen-activated protein kinase signaling cascades.

Interleukin-5 (IL-5) is a critical cytokine for the maturation of eosinophil precursors to eosinophils in the bone marrow and those eosinophils then accumulated in the lungs during asthma. We have studied anti IL-5 antibodies on allergic responses in mice, guinea pigs and monkeys and are extending this experiment into humans with a humanized antibody. In a monkey model of pulmonary inflammation and airway hyperreactivity, we found that the TRFK-5 antibody blocked both responses for three months following a single does of 0.3 mg/kg, i.v. This antibody also blocked lung eosinophilia in mice by inhibiting release from the bone marrow. To facilitate multiple dosing and to reduce immunogenicity in humans, we prepared Sch 55700, a humanized antibody against IL-5. Sch 55700 was also active against lung eosinophilia in allergic monkeys and mice and against pulmonary eosinophilia and airway hyperresponsiveness in guinea pigs. Furthermore, as opposed to steroids, Sch 55700 did not cause immunosuppression in guinea pigs. Studies with this antibody in humans will be critical to establishing the therapeutic potential of IL-5 inhibition.

A murine antibody to human tumour necrosis factor-alpha (TNF-alpha) (CB0010) was complementarity-determining region (CDR)-grafted using human IgG4 heavy and kappa light chain constant regions. In cynomolgus monkeys, the grafted antibody (CDP571) was eliminated with a half-life of 40-90 hr, two to three times longer than CB0010, and immunogenicity was reduced by > 90%. Responses to the constant regions were almost entirely eliminated and responses to the CDR loop (anti-idiotype) were lowered. CDP571 was given to 24 human volunteers in doses from 0.1 to 10.0 mg/kg. It was well tolerated, with a half-life of approximately 13 days. Anti-CDP571 antibodies were low or undetectable at higher doses. At lower doses, anti-CDP571 peaked at 2 weeks and then declined. The response was primarily IgM (in contrast to the cynomolgus monkey, where by 5 weeks IgG predominated) and was against a conformational epitope comprising heavy and light chain CDR loops. No antibodies were detected against the gamma 4/kappa domains or frameworks. The response had little or no effect on CDP571 binding to TNF-alpha or on plasma clearance.

Eosinophils infiltrate into the lungs during asthma and may cause the damage associated with pulmonary inflammation. In allergic animal models, antibodies to interleukin (IL)-5 inhibit pulmonary eosinophilia, tissue damage and hyperreactivity. Sch 55700, a humanized antibody against human IL-5, inhibits eosinophilia in these models with an extended biological duration. On the basis of this dosing regimen and the humanized nature of Sch 55700, it is anticipated that the host response leading to tolerance would be minimized.

Objective: To investigate the role of tumour necrosis factor alpha (TNF alpha) in the development of antigen induced arthritis (AIA) in rabbits., Methods: Monoclonal antibodies to rabbit TNF alpha were developed in rats and were used to detect TNF alpha in synovial fluid by enzyme linked immunosorbent assay and to localise it in tissue sections of synovium and cartilage from rabbits up to 21 days after induction of AIA. An antibody which neutralised TNF alpha activity in vitro was injected into rabbits to block TNF alpha action in vivo in AIA. Joint swelling, leucocyte infiltration into synovium and proteoglycan loss from cartilage were measured and compared with a control group, which were injected with sterile saline., Results: Monoclonal antibodies to purified rabbit TNF alpha were prepared in rats and two were selected which were able to neutralise rabbit TNF alpha in a cytotoxicity bioassay. TNF alpha was detected in significant concentrations (21.7 (SE 0.5) pg/ml) in the arthritic joint fluid of rabbits with AIA only at one day after induction and it was then also sparsely localised in cells of the synovium, but from day 3 onwards it was localised more strongly in the deep zone of articular cartilage. Injection of anti-TNF monoclonal antibody R6 over three days into rabbits with AIA reduced joint swelling and leucocyte infiltration into joint fluid and decreased the expression of CD11b and CD18 on cells in the joint fluid. However, there was no significant reduction in the loss of proteoglycan from articular cartilage, although the joint fluid at three days contained a lower glycosaminoglycan content. The antibody R6 gave most effect at a dose of 0.6 mg/kg and there was no increase in its effectiveness at a fivefold greater dose (3.0 mg/kg). Treatment over 10 days gave a more complete suppression of joint swelling, but did not result in any less proteoglycan loss from cartilage. Treatment for five days with a 16 day follow up gave a significant reduction in swelling for several days beyond the treatment, but the swelling then slowly returned, until by day 21 there was no significant difference in joint swelling and there was also no recovery of cartilage proteoglycan content. A rabbit anti-rat immunoglobulin response was detected at 21 days, which may have limited the long term effectiveness of the antibody., Conclusions: In AIA in rabbits, TNF alpha was only detected in synovial fluid at one day after induction and there was only limited cellular localisation of TNF alpha in synovium and cartilage from three days. However, neutralising TNF alpha with a monoclonal antibody was effective in suppressing inflammatory changes in the joint during the acute onset of AIA, but it had little effect on the loss of proteoglycan from cartilage. The results suggest that blocking inflammation and synovitis with anti-TNF alpha may be more easily achieved than preventing damage to articular cartilage.

Derivatives of plasmid pBR322 that are suitable for high-level expression of foreign genes have been constructed. The vectors contain the Escherichia coli tryptophan promoter, the trpE gene, and about 15% of the trpD gene. To obtain expression, foreign genes are fused to the trpD gene fragment. After induction of the trp operon with 3 beta-indolylacrylic acid, trp gene products increase at least 50-fold, to account for 55% of the newly synthesised protein and 30% of total protein in the cell.

A DNA fragment flanked in the E. coli genome by Hinf I sites and containing the E. coli tryptophan promoter-operator and nucleotides specifying the leader sequence and first seven amino acids of trp E, has been cloned in the Hind III site of pBR322 with the aid of Hind III linkers. The Hind III site upstream from the trp promoter in the transcriptional sense was deleted to generate a Hind III cloning vector, designated pWT111, suitable for the expression of cloned DNA sequences as fusion products. Derivatives of this plasmid were constructed, designated pWT121 and pWT131, whose Hind III cloning sites differ with respect to their translation phasing relative to the initiator ATG of the trp E gene. Both pWT121 and pWT131 have a higher copy number per cell than pWT111. The tetracycline genes of all three plasmids are under trp promoter control and could be used to monitor the transcription of foreign genes inserted at the Hind III site of the vectors.

The complete sequence of a hemagglutinin (HA) gene of a recent human influenza A strain, A/Victoria/3/75, is 1768 nucleotides long and contains the information for 567 amino acids. It codes for a signal peptide of 16 amino acids, the HA1 chain of the mature hemagglutinin of 329 amino acids, a connecting region between HA1 and HA2 consisting of a single arginine residue and the HA2 portion of 221 amiino acids. The sequence is compared with the hemagglutinin of two members of other subtypes, the human H2 strain A/Jap/305/57 and the avian Hav1 strain A/FPV/Rostock/34, and with one of the same H3 subtype, A/Memphis/3/72. To align the HA1 chain of different major subtypes several deletions/insertions of single amino acids must be invoked, but two more extensive differences are found at both ends, one leading to an extension of the amino terminal sequence of HA1 and the other (four residues) occurring in the region processed away between HA1 and HA2. Comparison of the HA1 of two H3 strains suggests that drift probably depends on single base mutations, some of which change antigenic determinants. The HA2 region, which apparently is not involved in the immune response, is highly conserved even between different subtypes, and single base substitutions account for all the observed diversity. A hydrophobic segment of 24 residues is present in the same position close to the carboxyl terminus of HA2 in both Victoria and FPV, and presumably functions in implantation into the lipid bilayer. The many conserved features not only in HA2 but also in HA1 suggest a rather rigid architecture for the whole hemagglutinin molecule.